Cellular transport system for the transfer of a nucleic acid through the nuclear envelope and methods thereof

ABSTRACT

The present invention relates to a nuclear transport agent, to a gene transfer system comprising said nuclear transport agent, to a method for transporting DNA into the nucleus of eukaryotic cells using said nuclear transport agent and to the use of said nuclear transport agent in gene therapy for treating cancer, viral infections, diseases of the nervous system, graft rejection and monogenic or polygenic hereditary diseases.

This application claims benefit of International Application No.PCT/DE00/00061, filed Jan. 3, 2000; which claims priority of GermanApplications No. 199 33 939.2, filed on Jul. 20, 1999 and 199 00 513.3filed on Jan. 8, 1999. The contents of all of the foregoing applicationsin their entireties are incorporated by reference into the presentapplication.

The present invention relates to a nuclear transport agent, to a genetransfer system comprising said nuclear transport agent, to a method fortransporting DNA into the nucleus of eukaryotic cells using said nucleartransport agent and to the use of said nuclear transport agent in genetherapy for treating cancer, viral infections, diseases of the nervoussystem, graft rejection and monogenic or polygenic hereditary diseases.

The active transport into the nucleus is necessary for the transfer ofgenetic material into all cells that do not divide in the period beforethe intended expression of the genetic material. A nuclear transportsystem for nucleic acids is very important because it facilitates theefficient transfer of DNA into those cells that divide rarely or not atall (Dowty et al., 1995, Wilke et al., 1996). Most primary cells belongto this group. Primary cells are of highest scientific interest for tworeasons. Firstly, said cells that have freshly been isolated from anorganism reflect the functional state of the cell type much better thancell lines derived therefrom. Secondly, they are the target cells forgene therapy. In addition, a nuclear transport system increases theefficiency of DNA transfer into established cell lines by enabling alsothose cells to express transferred genetic material, which have notdivided in the period of time between start of transfer and analysis.

Genetic material is active in the nucleus. The transport therein caneither occur coincidentally during cell division when the nuclearenvelope temporarily disintegrates in the course of mitosis or it has totake place actively.

1) Nuclear Proteins are Transported into the Nucleus by Means of NuclearLocalization Signals

The double membrane that envelops the nucleus has pores. Littlemolecules can pass through these pores by diffusion. In order to be ableto enter the nucleus, proteins larger than about 50 kDa need a nuclearlocalization signal (NLS) that has to be recognized by the transportmachinery. Typically, a sufficient signal consists of four to eightamino acids, is rich in the positive amino acids arginine and lysine andcontains prolines. It is strongly conserved in evolution so thatmammalian NLS are also functional in yeast. Heterologous NLS can also beused as a tool to transport target molecules into the nucleus. For thispurpose, NLS can be incorporated into the sequences of cytoplasmicproteins at relatively random positions or can be coupled chemically toproteins or even gold particles (reviewed in Görlich, 1998).

2) Many Viruses Use Nuclear Protein Transport Machinery of the Cell forthe Transport of Their DNA into the Nucleus

HIV and other lentiviruses that are able to infect resting cells useviral proteins and the cellular transport machinery to transfer theirDNA into the nucleus. The NLS in Vpr and matrix protein of the HIVpre-integration complex (Gallay et al., 1996) are essential for theinfection of cells that do not divide (Naldini et al., 1996). Althoughlittle is known about how viruses transfer their genomes into thenucleus, the help of viral structural proteins containing NLS might evenbe a general principle. This is also suggested by the followingobservations: A specific mutation in the HSV capsid protein prevents thetransport of viral DNA into the nucleus (Batterson et al., 1983).Adenovirus DNA is transported into the nucleus together with the hexonprotein of the disintegrated capsid (Greber et al., 1993). The transportof SV40 DNA into the nucleus is mediated by a viral protein (probablyVp3) that remains associated with DNA (Nakanishi et al., 1996). Twobacterial proteins containing NLS are responsible for the import ofAgrobacterium tumefaciens T-DNA into plant nuclei (Citovsky et al.,1994).

Due to the ability of some viruses to infect resting cells, mutantvariants of, for example, HIV, adenovirus and herpes virus are used asDNA transfer vehicles for the development of gene therapy approaches.Firstly, this involves the risk of immunological reactions to viruscomponents (Friedmann, 1994, 1996) and, secondly, helper cell lines areused in such systems for which the release of less mutated virus genomescannot be excluded. Moreover, the handling of these systems isdifficult.

Several artificial systems have been described that are supposed toincrease transfection efficiency by means of peptides or proteinscontaining nuclear localization signals.

A) Proteins

Kaneda et al. (1989) and Dzau and Kaneda (1997, U.S. Pat. No. 5,631,237)describe a gene transfer system that is based on the use of Sendaivirus, liposomes and added proteins that are meant to support nucleartransport of DNA. For this purpose, the group used HMG-1 (high mobilitygroup 1 protein), a basic non histone protein of chromatin which bindsto DNA. HMG-1 binds to DNA through a long basic region. It is localizedin the nucleus, but does not have a known NLS. In vitro, HMG-1 proteinforms complexes with vector DNA. The production of purified HMG-1 iscostly and labor-intensive.

Mistry et al. (1997) describe experiments concerning HMG-1-mediatednuclear transport. Due to its positive charge HMG-1, as a transfectionreagent that complexes DNA, is used here for the passage of DNA throughthe cell membrane. The efficiency is low. The company Wako BioProducts(Richmond, Va., U.S.A.) sold (1997) the proteins HMG-1 and -2 asadditives for lipofection reagents to mediate nuclear transport.

Fritz et al. (1996) followed a similar approach with calf thymushistones or a recombinant protein consisting of SV40 NLS and humanhistone H1. Both of these proteins evidently form large complexes withDNA, as was shown in the publication, and are suitable for the passageof the cellular membrane but not for nuclear transport.

B) Due to Their Simpler and Less Expensive Production, SyntheticPeptides Containing NLS Sequences Were Used as Well

The group of P. Aleström (Collas et al., 1996, Collas and Alestrom,1996, 1997a, b) uses the NLS peptide from SV40 to complex DNA and haveit transported into the nucleus by the cell. This DNA binding occurssolely through the positively charged amino acids of the NLS that areessential for its function. This results in masking the actual signalfor the nuclear transport proteins as long as the DNA is complexed withthe peptide. An NLS-dependent transport of fluorescently labeled DNAcould be observed in isolated male pronuclei formed in vitro from seaurchin sperms, when they have been incubated in the lysate of fertilizedzebra fish eggs. At a molecular ratio of ≧100:1 (NLS peptide:vector) and≧1,000 vector copies per cell, an increase in luciferase expressioncould be observed in zebra fish embryos, when vector DNA wasmicro-injected. into the cytoplasm of the cells. (At 100 peptides/vectorand 1,000 injected vectors a sixfold increase was obtained as comparedto 0 peptide.) Due to the high density, possibly not all NLS bindcompletely to the DNA and thus parts remain accessible for the transportmachinery; this might be the cause why an effect can be perceived at all(cf. Sebastyén et al., 1998). The transport machinery is probably ableto recognize signals composed of two peptide sequences (Boulikas, 1993).

Sebastyén et al. (1998) covalently coupled many hundreds of SV40 NLSpeptides to DNA molecules, with the NLS being scattered over the entirelength of the DNA strand. Due to its massive modification, the DNA canno longer be transcribed. As is discussed in the article, the DNA isevidently only transported into the nucleus when so many NLS peptidesare bound that, for steric reasons, not all of them are masked by theinteraction with the negative charges of the DNA.

Gopal (U.S. Pat. No. 5,670,347) describes a peptide that consists of aDNA-binding basic region, a flexible hinge region and an NLS. As DNAbinding is also in this case achieved by the amino acids' positivecharges, the reagent forms complexes with the DNA that are meant toserve at the same time for the transport across the cellular membrane.It is not evident why the NLS sequence should not participate in thebinding of DNA so that the actual signal for the nuclear transportproteins is again likely masked by the DNA as long as the peptide iscoupled thereto. Moreover, the complexes generated may become very large(Emi et al., 1997, Niidome et al., 1997, Wadhwa et al., 1997, Trubetskoyet al., 1998), which would impair transport through the nuclear pores(Lanford et al., 1986, Yoneda et al., 1987, 1992). An effect beyond theknown function of polycationic peptides as a transfection reagent, whichsupports the passage of DNA through the cellular membrane (Sorgi et al.,1997, cf. Hawley-Nelson et al., 1997) has not been shown.

Gerhard et al. (DE-OS 195 41 679) suggest NLS polylysine conjugates forgene transfer. It is also true in this case, that the emerging complexesconsisting of cationic polylysine, cationic NLS and DNA mask the nucleartransport signal as long as it is coupled to the DNA.

Szoka (PCT 1993, claims 23-27) couples NLS peptides to DNA via anintercalating agent. After pre-incubating vector and peptide (ratio of1:300), the efficiency of lipofection increases four- to fivefold. Dueto its highly positive charge, the SV40 peptide used is able to complexDNA. Complexing of DNA with cationic peptides leads to an increasedlipofection efficiency by improving the efficiency of passage across thecellular. membrane (Sorgi et al., 1997, cf. Hawley-Nelson et al., 1997).Nuclear transport is rather impaired thereby, at least when largecomplexes are generated (see above). As the NLS peptides used bind toDNA due to their charge, the recognition of the transport signal by thenuclear transport machinery is impaired (see above). The use ofmutagenetic intercalators described in the example restricts theapplicability. Szoka suggests additional molecules for transfection thatalso bind to DNA non-covalently and unspecifically but, as before,cannot prevent the NLS peptide itself from binding to and complexing theDNA. The problem of a direct association of the NLS peptide with the DNAis not discussed.

Hawley-Nelson et al. (U.S. Pat. No. 5,736,392) describe a similarsystem. An NLS peptide is mixed with vector DNA either directly or aftercovalent coupling to a DNA-binding molecule. The complexes generated arethen used for lipofection (or other transfections). In this system theaddition of a polycationic peptide without NLS increases thetransfection efficiency even more than the addition of a cationic NLS.The coupling of spermidine to the NLS peptide does result in a furtherincrease in transfection efficiency. Thus, also in this case, theamplification effect is solely explained by the complexing of DNA viacationic peptides. As the presence of NLS does not increase thetransfection efficiency any further, it is to be assumed that therecognition sequence for the nuclear transport machinery is masked inthis case, as well.

The company TIB Molbiol (leaflet 1998) describes the transport of PNAoligonucleotides with a C-terminal NLS peptide to specifically suppressthe expression of selected genes. The NLS serves for the transport ofthe PNA oligonucleotides into the nucleus so that they can thenhybridize with their target sequence.

So far, the known agents for the transport of DNA into the nucleus havethe disadvantage that the efficiency is very low. This low efficiency isinsufficient to render resting cells transfectable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of PNA-NLS on the transfection efficiencies aspercent of transfected cells. Transfection of CHO cells with or withoutPNA-NLS and in the presence or absence of Aphidicholin, as described inExample 3, is shown.

FIG. 2 shows an autoradiograph of a DNA-retardation gel of a DNA-bindingassay with mutated lac-repressor protein containing different NLS, asdescribed in Example 5.

FIG. 3 is a fluorescent microscopic representation of NIH3T3-cells,microinjected into the cytoplasm with fluorescently labeled DNA with orwithout lac-repressor-NLS protein, as described in Example 6.

FIG. 4 is a graphic representation of transfection efficiencies with orwithout lac-repressor-NLS protein, shown as percent of transfectedNIH3T3-cells, as described in Example 7.

DESCRIPTION OF THE INVENTION

Thus, the problem underlying the present invention is to provide anuclear transport agent that facilitates the efficient transport of DNAinto the nucleus so that also resting or only very slowly dividing cellsbecome transfectable to a useful degree.

This problem is solved by a nuclear transport agent consisting of twomodules A and B, where module A binds specifically to DNA and does notlead to the formation of complexes containing more than one DNA moleculeby unspecific binding, and where module B contains a nuclearlocalization signal or a non NLS signal that does not bind to DNAunspecifically. A preferred nuclear transport agent according to thepresent invention comprises a module A that binds sequence specificallyto DNA and/or binds specifically to DNA ends. Particularly preferred isa nuclear transport agent where module A is a synthetic peptide, aprotein or a peptide nucleic acid (PNA).

In a further embodiment of the nuclear transport agent according to thepresent invention, module B contains an extended nuclear localizationsignal that does not form complexes with DNA due to its charges. Anuclear transport agent is preferred in which module B contains anextended nuclear localization signal that possesses an approximatelyneutral net charge. A nuclear transport agent is particularly preferredin which module B contains an extended nuclear localization signal thatcomprises a nuclear localization signal and flanking negatively chargedamino acids. A NLS sequence does not have to be identical to a naturallyoccurring NLS sequence but can also be an amino acid sequence based ontheoretical consideration as long as it is functional as NLS. Moreover,module B can contain peptide sequences or non-peptide components that donot directly belong to the nuclear localization signal or extendednuclear localization signal. Preferred is a component that increases thedistance between the nuclear localization signal and module A.

Moreover, the invention concerns a gene transfer system comprising anuclear transport agent according to the present invention and acationic lipid, peptide, polyamine or cationic polymer.

Moreover, the invention concerns a method for the transport of DNA intothe nucleus of eukaryotic cells, preferably primary cells, wherein thecells are transfected with the DNA to be transported and the nucleartransport agent according to the present invention by methods known inthe art.

A further embodiment concerns the use of the nuclear localization agentaccording to the present invention in gene therapy, in particular forthe treatment of cancer, viral infections, diseases of the nervoussystem, graft rejection as well as monogenic or polygenic hereditarydiseases.

The expression “unspecific binding of the nuclear localization signal toDNA”, as used in the present invention, denotes an association thatprevents the nuclear localization signal from being completelyrecognizable to the nuclear transport machinery.

The expression “specific binding of module A to DNA”, as is used in thepresent invention, denotes, firstly, sequence-specific binding, in whichthe sequence of DNA nucleotides is crucial for the interaction and,secondly, a covalent binding with DNA that is mediated by DNA single ordouble strand ends.

The expression “extended nuclear localization signal”, as is used in thepresent invention, denotes that a nuclear localization signal possessesadditional flanking amino acids. Preferred is an extended nuclearlocalization signal that possesses 2 to 40, preferably 4 to 20,additional flanking amino acids.

The expression “extended nuclear localization signal that does not formcomplexes with DNA due to its charge”, as is used in the presentinvention, denotes that module B contains a nuclear localization signalwhose charges are distributed in such a way that it does not interactwith DNA unspecifically and thus remains completely accessible for thenuclear transport machinery.

The expression “approximately neutral net charge”, as is used in thepresent invention, denotes that the extended part of the nuclearlocalization signal possesses negatively charged amino acids to balancethe positive charge of the actual nuclear localization signal so that nomore than three positive surplus charges occur in the entire region ofthe extended nuclear localization signal.

The nuclear transport agent according to the present invention has theadvantage that it does not lead to complexing of DNA. It is a furtheradvantage that the nuclear localization signal remains freely accessibleto the nuclear transport machinery. Avoiding large DNA complexes thatimpair nuclear transport and the accessibility of the nuclearlocalization signals to the nuclear transport machinery when using thenuclear transport agents according to the present invention, leads to aclearly more efficient transport of DNA into the nucleus.

According to the present invention neither the DNA-binding part (moduleA) nor the nuclear localization signal (module B) leads to the formationof large DNA complexes.

Module A

Module A binds specifically to DNA and does not lead to the formation ofcomplexes with more than one DNA molecule. Module A binds eithersequence specifically (i.e. not unspecifically only due to positivecharges) or covalently to DNA ends.

Module A can be a peptide of varying lengths or a protein or a PNAsequence (Nielson et al., 1991) or another substance that binds tonucleic acids in a sequence-specific manner. Moreover, module A can be arecombinant protein that binds to DNA specifically, as for example lacrepressor or a high-affinity mutant thereof (Kolkhof, 1992, Fieck etal., 1992), or a retroviral integrase that binds sequence specificallyto DNA ends (with an LTR core sequence) (Ellison and Brown, 1994).

Covalent binding to the end of a DNA strand can be mediatedbiologically, for example by topoisomerase I of the poxvirus, if the endof a linear DNA strand has a sequence that is a “suicide substrate” andpermits cleavage by topoisomerase but no relegation (Shuman, 1994).

Module B

Module B is a nuclear localization signal or a non NLS signal that doesnot bind unspecifically to DNA.

The term non NLS signals according to the present invention denotessignals which are not nuclear localization signals but with regard totransfection, gene therapy or DNA vaccination serve to transport the DNAinto the cell or to transport DNA within the cell.

The following belongs to non NLS signal: ligands for cellular surfacestructures, which are able to mediate DNA uptake, e.g. receptor mediatedDNA uptake; peptides which destabilize membranes, e.g. to promotepremature exit of DNA from endosomes; signals mediating in the cellbinding to transport structures to favor intracellular transport to thenucleus.

The nuclear localization signal is preferably an extended nuclearlocalization signal (as defined above) that does not form complexes withDNA due to its charge or spatial orientation to DNA binding module A.The nuclear localization signal can be generated synthetically or can bepart of a protein.

In a nuclear localization signal as is used in the nuclear transportagent according to the present invention, signal sequences—with andwithout flanking regions—are used that do not bind to DNA via theirpositive charges in such a way that these charges which are an essentialpart of most nuclear localization signals, are masked as signals for thenuclear transport machinery.

Apart from the nuclear localization signal or non NLS signal, module Bmay contain peptide sequences and non-peptide components that are notpart of the nuclear localization signal or the extended nuclearlocalization signal. Preferably, they permit a better steric positioningof the nuclear localization signal, especially an increased distance tothe DNA molecule.

Extended sequences of classic NLS are well suited provided that thepeptide's net charge can almost be balanced by flanking negativelycharged amino acids. These amino acids can occur naturally at thesepositions in the protein or may have been introduced on the basis ofstructural considerations. In the original context, negative amino acidsare located adjacent to many NLS core sequences (Xiao et al., 1997). Itcould be shown for an NLS from SV40 which is the most thoroughlyinvestigated NLS, that these flanking sequences clearly increase theefficiency of nuclear transport (Rihs and Peters, 1989, Rihs et al.,1991, Chen et al., 1991, Jans et al., 1991, Xiao et al., 1997). A largeprotein (IgM) was transported into the nucleus only after coupling itchemically to the SV40 NLS that had been extended by adjacent sequences,but not after coupling it to the SV40 core NLS peptide (Yoneda et al.,1992).

If the NLS is part of a protein that binds sequence specifically to DNA,the risk with these sequence(s) of being masked by DNA is relativelylow. But due to the higher efficiency, extended NLS sequences can alsobe used in this case.

Non-classic NLS, as for example the NLS from the influenza virusnucleoprotein (Wang et al., 1997, Neumann et al., 1997) which do nothave a large excess of positive charges or do not reach the nucleus viathe conventional route of transport can also be used.

An (incomplete) overview of NLS as they are intended here is given by T.Boulikas (1993, 1996, 1997).

Finally, nuclear transport signals can be used that are taken fromcomponents of the nuclear transport machinery itself, as for example theimportin β binding domain (IBB) of importin α. Via this domain, the NLSbinding protein importin α is linked to the rest of the nucleartransport machinery (Görlich et al., 1996, Weiss et al., 1996).

In a further preferred embodiment a non NLS signal via PNA (as module A)is bound to existing vector sequences. The binding between PNA andvector is sequence specific. This allows coupling of such non NLSsignals to almost all conventional expression vectors without the needto modify them.

For sequence specific binding of PNA to the DNA only those DNA sequencesof the vector are used, the masking of which by PNA does notsubstantially impair the intended purpose of the DNA.

In the case of expression vectors, in particular sequences in theplasmid backbone are used especially those which are present in mostconventional expression vectors (e.g. promoter of ampicillin resistancegene). However, also binding in the non coding strand of the expressionregion is possible. An advantage of the sequence specific binding is asimple and rapid binding of the PNA-peptide-hybrid to the DNA. Example 2demonstrates for example a simple and rapid binding reaction (5 min, 65°C.) of PNA-peptide-hybrids to double stranded DNA. There may be a spacerbetween the PNA portion and the actual signal. The spacer may serve toincrease the distance of signal to DNA, e.g. to reduce steric hindrance.The spacer may also serve to introduce a predetermined breaking point,e.g. to allow the separation of a ligand in the endosomal milieu, viawhich the DNA is bound to an endocytosed cell surface receptor.

For the first time, the present invention renders resting or slowlydividing cells transfectably to a percentage that allows subsequentanalysis. Most cells freshly isolated from the body of an animal orhuman (primary cells) do not divide at all or so rarely that DNA, afterit has been transported across the cellular membrane successfully, isinactivated before it reaches the nucleus and can be expressed. So farthis has led to primary cells being untransfectable unless they wereartificially stimulated to proliferate in culture. The unavoidableconsequence of this is that these cells then deviate from their originalstate. A method for the transfection of primary cells permits theanalysis of genetic material under the original conditions of a bodycell. This is of paramount importance for the investigation of geneticmechanisms and the study of processes inside a body cell.

The teaching according to the present invention that renders primarycells transfectable is also an essential step toward a completelyartificial gene transfer system for gene therapy. Such a gene transfersystem must have three functional components: one component for thepassage of DNA through the cellular membrane, for which cationic lipidsand other cationic polymers have proved to be relatively suitable. Ithas to contain a further component for the transfer of the DNA into thenucleus of the (usually non-dividing) target cells and a third componentthat mediates the integration of the DNA into the genome. In the presentinvention for the first time an efficient agent is described that canserve as the second component. A completely artificial gene transfervehicle that can be employed in gene therapy is expected to be producedeasier and less expensive and handled easier than the viral systemscurrently used, and it is not subject to the immanent risks of thesesystems. Gene therapeutic approaches have been suggested, for example,for the treatment of cancer, AIDS and various hereditary diseases andwill play a significant role in medicine.

The nuclear transport agents described according to the presentinvention also increase the transfection efficiency in such culturedcells that up to now have already been transfectable by making thosecells accessible to the uptake of DNA that do not divide in the periodbetween the passage of the DNA through the cellular membrane andanalysis. This is important because even for many established cell linesan increase in transfection efficiency would facilitate the analysis andhelp lower costs due to the reduced amount of cell material required. Ofcourse, this is also true for all stages in between primary cells andestablished cell lines.

The following examples illustrate the invention and are not intended tolimit the scope thereof.

EXAMPLE 1

PNA-peptide-hybrids

NLS PNA nuclear transport agents were used. PNA sequences were used thatare capable of invading DNA double strands (Nielson et al., 1991,Nielson, U.S. Pat. No. 5,539,082).

In the plasmid backbone of almost all expression vectors, two sequencesthat are well suited for a high-affinity association with PNA arelocated in the ampicillin resistance gene and the origin of replication.

As peptide components the peptides employed contain: either

1) “SV21” NH₂-GKPTADDQHSTPPKKKRKVED-COOH (peptide 1, SEQ ID NO:1), or

2) “SV27” NH₂-GKPSSDDEATADSQHSTPPKKKRKVED-COOH (peptide 2; SEQ ID NO:2).

The following PNA sequence is located at the N-terminus of each peptide.Either

A) “ori” NH₂-CCTTTCTCCCTTC-peptide (SEQ ID NO:3), or

B) “ssp” NH₂-CTCTTCCTTTTTC-peptide (SEQ ID NO:4), or

C) the peptide-PNA hybrid sequence NH₂-CCTTT-GGGGGGG-TTTCC-peptide(CCTTT (SEQ ID NO:5); GGGGGGG (SEQ ID NO:10); TTTCC (SEQ ID NO:11)) thathas about 30 binding sites in an average expression vector (5 kb) (G=theamino acid glycine).

5 μg of vector DNA solubilized in water were incubated in 10 μl 25 μMNLS-PNA for 10 min at 60° C. The reaction mixture was then adjusted to250 μl with RPMI.

5×10⁶ Chinese hamster ovary (CHO) cells that were 60 to 80% confluentwere detached with 5 mM EDTA, washed in 15 ml PBS (centrifuged at 50×gfor 10 min). The pellet was resuspended in 250 μl RPMI and mixed withthe pre-incubated DNA, transferred to an electroporation cuvette (gapwidth of 0.4 cm) and incubated at room temperature for 10 min. Afterelectroporation (210 V, 975 μF, BioRad GenePulser) the cuvette wasincubated at 37° C. for another 10 min before the cells were seeded inpre-warmed medium.

In order to show unequivocally that those cells were transfected thathad not divided between the start of the experiment and analysis, cellswere transfected with pMACS 4.1 (an expression vector for human CD4)according to the method described and cell division was assessed asfollows: Before transfection, cells were labeled with green fluorescenceby incubation in 1 μM carboxyfluorescein diacetate succinimide ester(CFDA, SE) (Molecular Probes, Eugene, U.S.A.). The brightness of thecells is reduced to 50% by cell division. Using a flow cytometer(FACSCalibur), it was determined on the single-cell level that alsocells that had not divided (100% green fluorescence) expressed thetransfected gene (dark red fluorescence after staining with anti-CD4antibody coupled to Cy5).

EXAMPLE 2

Rapid Binding of PNA-NLS to Existing Vector Sequences

PNA-peptide-hybrids were coupled to double stranded DNA.

An existing vector sequences can be labelled via PNA almostquantitatively (>90%) with an NLS-peptide within 5 minutes. To achievebinding of heat labile components via PNA, incubating for one hour at37° C. is sufficient to label most of the DNA (Table 1).

100 ng expression vector were incubated in TE (pH 7,8) with 25 μM ofdifferent PNA-peptide-hybrids at either 65° C. or 37° C. for fiveminutes to three hours. The PNA-sequence NH₂-CTCTTCCTTTTTC-COOH (SEQ IDNO: 6) used here, binds to the promoter of the ampicillin resistancegene.

At the C-terminus either a peptide of 21 amino acids (Peptide 1) or 27amino acids (Peptide 2) or a spacer of 10 AEEA-units (Fmoc-AEEA-OHSpacer, PerSeptive Biosystems Inc., Framingham, USA) followed by 27amino acids (Peptide 3) is located. The binding assays were subsequentlyincubated with restriction endonuclease Earl. Restricted DNA was stainedwith YOYO (Molecular Probes, Inc., Eugene, Oreg., USA) separated on anagarose gel and quantified with a fluorescence scanner (Image PlateReader FLA 2000, analysis software L-Process, version 1.6, Fuji PhotoFilm Co., Ltd., Tokio). Cleavage of DNA by restriction endonuclease Earlis inhibited at the PNA binding site. Additional Earl restriction sitesserve as internal control of the reaction.

TABLE 1 Portion of peptide-labelled DNA shown as percentage of input DNA° C. min. Peptide 1 Peptide 2 Peptide 3 65  5 94% 96% 91% 10 96% 97% 95%15 96% 97% 95% 37 60 85% 90% 74% 120  91% 91% 76% 180  94% 94% 90%

The binding reaction of PNA to DNA is simple, robust and rapid. Thebinding is almost irreversible and therefore suitable for cellulartransport processes. Compared to proteins, peptides and PNA can besynthesised less expensively and can be stored easier and for a longertime.

EXAMPLE 3

Transfection Using PNA-NLS

In dividing cell lines active nuclear transport of transfected DNAprovokes its sooner expression compared to DNA which is not transported.Provided that the transfected DNA survives in the cytoplasm for a timelong enough, the expression rates of transfected DNA in rapidly dividingcells with and without nuclear transport reagent should approximatelittle by little. The reason for this is the fact that transfected DNAthat remains in the cytoplasm can reach the nucleus during celldivision. Using aphidicolin the division activity and the transfectionability of cells can be strongly reduced. Active nuclear transportabolishes this effect of reduced transfection efficiency.

For electroporation 5 μg of linearized vector-DNA, dissolved in water,were incubated in a final volume of 10 μl with or without 25 μM PNA-NLS(Peptide 3: NH₂-(AEEA)₁₀-GKPSSDDEATADSQHSTPPKKKRKVED-COOH; (SEQ IDNO:7)) for 10 min at 65° C. The further procedure was as described inexample 1.

For lipofection 3 μg of vector-DNA, dissolved in water, were incubatedin a final volume of 10 μl with or without 25 μM PNA-NLS (Peptide 3) for10 min at 65° C. Transfection with lipofectamine (Life TechnologiesGmbH, Karlsruhe) was done according to the manufacturer's instructions.

To inhibit division of CHO cells substantially, cells were incubatedwithout serum for 24 hours followed by a 12-hour-incubation with serumand 20 μg/ml aphidicolin (Sigma-Aldrich Chemie GmbH, Deisenhofen). Allsubsequent steps of lipofection were done in the presence of 20 μg/mlaphidicolin. The results of transfection are shown in FIG. 1.

Two electrically neutral NLS, which are coupled to a sequence present inmost of the expression vectors are capable of duplicating the percentageof transfected cells early after transfection although only a few cellshave divided. Reduction of transfection efficiency caused by thereduction of cell division rate using aphidicolin can be abolished inthis way.

EXAMPLE 4

Sequence-specific-binding NLS-fusionprotein

A high-affinity binding mutant of the E.coli lac repressor was used assequence-specific DNA-binding protein. This mutant has a bindingconstant of 10⁻¹⁵ M for the lac operator sequence (Kolkhof, 1992). Thehigh affinity is achieved by an amino acid replacement of serine 61 toleucine.

The nuclear transport proteins used here have a deletion of the lastthirty C-terminal amino acids (position 331-360) and a replacement ofleucine at position 330 to serine. These mutant proteins form homodimersinstead of homotetramers and therefore contain one single DNA-bindingsite instead of two sites. But also tetramers may be used as nucleartransport agent of the invention.

The dimer-variants each were extended at the N-terminus for one NLS:

“N1D” NLS1: MPKKKRKV-MKPVTLYDVA . . .

“N2D” NLS2: MEEDTPPKKKRKVEDL-KPVTLYDVA . . .

The NLS-sequences are shown in bold and correspond to SEQ ID NO: 8 andSEQ ID NO: 9, respectively. Sequences MKPVTLYDVA (SEQ ID NO:12) . . .and KPVTLYDVA (SEQ ID NO:13) . . . indicate the E.coli lac repressorspecified above.)

NLS1 corresponds to the NLS of the SV40 virus large T antigen. NLS2represents a hybrid with neutral net charge consisting of the SV40-NLSand the N-terminal flanking region of the NLS from Polyoma virusVP2-protein.

Lac-operator-sequences can be found in a number of expression vectorsand can easily be joined to any sequence as extensions of PCR primers.

EXAMPLE 5

DNA-binding of Lac-repressor Mutants Containing NLS

The following lac-operator-sequences were used for binding assays:

The naturally occurring operator: AATTGTGAGC GGATAACAATT (SEQ ID NO:14)and a perfectly palindromic operator-sequence: AATTGTGAGC GCTCACAATT(SEQ ID NO: 15).

0.7 ng of a radioactively labelled DNA-fragment of 1 kb length wascleaved by restriction endonucleolytic digestion into fragments of 914bp and 86 bp length and then incubated for 30 min at room temperaturewith lac-repressor NLS-1-dimer or NLS-2-dimer, respectively. Thefragments were then separated on a polyacrylamide gel (FIG. 2). The 86bp-fragment, which contains the lac-operator, is retarded, due tospecific binding. Non-specific binding results in retardation of the 914bp-fragment lacking the lac-operator. In the case of complete specificbinding hardly any non-specific binding is observed.

Further experiments demonstrated, that stable binding is achieved usingvarious conditions e.g. in cell culture medium RPMI, 150 mM sodiumchloride or a buffer consisting of 10 mM Tris/HCI (pH 7.2), 10 mMpotassium chloride and 3 mM magnesium acetate with both testedoperator-sequences.

EXAMPLE 6

Nuclear Transport of DNA by Lac-repressor-NLS

Approximately 8 μg (100 pmol) lac-repressor-NLS-mutants were incubatedwith 2 μg (100 pmol) double-stranded DNA of 30 bp length, labeled atboth ends with the fluorescent dye Cy5, for 30 min at room temperaturein a total volume of 300 μl 10 mM Tris/HCl (pH 7.2), 10 mM KCl, 3 mMMg-Acetate and 50 μg/ml BSA. Unbound DNA was separated by centrifugationthrough a Microcon-filter (Amicon). The buffer of the sample was thenchanged to cell injection buffer (76 mM K₂HPO₄, 17 mM KH₂PO₄, 14 mMNaH₂PO₄ (pH 7.2)).

A mixture, consisting of DNA, bound to lac-repressor-mutants, andfluorescein-labeled BSA (BSA-FITC) was microinjected (EppendorfTransjektor 5246 with Femtotips, diameter 0.5 μm, pressure of injection55 hPa, time of injection 0.5 sec) into 50 NIH3T3-cells, respectively.Ten to 15 min after injection cells were analysed by fluorescencemicroscopy (FIG. 3). Following successful injection into the cytoplasm,BSA-FITC resides exclusively in the cytoplasm (1a, 2a, and 3a). Bindingto lac-repressor-NLS-mutants results in nearly all cells, which could beanalysed, in transport of the DNA into the nucleus within less than 15min (NLS1-Dimer, 2b) and less than 10 min (NLS2-Dimer, 3 b),respectively, leaving nearly no DNA in the cytoplasm. Controlsdemonstrate that labeled DNA without binding proteins remains in thecytoplasm (1b).

EXAMPLE 7

Transfection with Lac-repressor-NLS

One microgram of a linear DNA of 1.1 kb length, containing a completeexpression-cassette and a polyadenylation sequence followed by a perfectpalindromic lac-operator-sequence, was incubated in 50 μl isotonic0.5×RPMI (for lipofection) or 150 mM NaCl (for transfection usingpolyethylenimine) for 30 min at room temperature with differentconcentrations of lac-repressor-protein (approx. 2.5 μg, 0.3 μg and 0.15μg dimer, and 5 μg, 0.6 μg or 0.3 μg tetramer, respectively). Thesamples were complexed with Lipofectamine (Life Technologies) orPolyethylenimine (PEI, ExGen 500, Fermentas) according to themanufacturer's instructions and added to confluent NIH3T3-cells. Theresults are shown in FIG. 4.

Transfection efficiency, determined 4 hours past transfection, can beincreased by the lac-repressor-NLS by a factor of 3-4. In the exampledescribed here, adherent NIH3T3 cells were cultivated to confluencebefore transfection, leading to an extensive inhibition of celldivision. Four hours past transfection only a few cells have divided.The period in which the anyway limited division rate in this examplebecomes relevant for transfection, is additionally reduced by the factthat transfected DNA, which is taken up by endocytosis, has to leave theendosomes and subsequently the complex with the cationic transfectionreagent, before it can be transported to the nucleus to be expressed.Lipofectamine-DNA-complexes probably persist noticeably longer thanDNA-complexes with polyethylenimine, leading to a less clear effect oflac-represssor-NLS using Lipofectamine. After most of the cells havedivided once 24 hours later, the expression rates of transfected DNAwith and without a nuclear transport reagent approximate gradually.

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15 1 21 PRT Artificial Sequence SV21 1 Gly Lys Pro Thr Ala Asp Asp GlnHis Ser Thr Pro Pro Lys Lys Lys 1 5 10 15 Arg Lys Val Glu Asp 20 2 27PRT Artificial Sequence SV27 2 Gly Lys Pro Ser Ser Asp Asp Glu Ala ThrAla Asp Ser Gln His Ser 1 5 10 15 Thr Pro Pro Lys Lys Lys Arg Lys ValGlu Asp 20 25 3 13 DNA Artificial Sequence DNA is PNA (peptide nucleicacid) 3 cctttctccc ttc 13 4 13 DNA Artificial Sequence DNA is PNA(peptide nucleic acid) 4 ctcttccttt ttc 13 5 5 DNA Artificial SequenceDNA is PNA; mixed peptide/PNA sequence 5 ccttt 5 6 13 DNA ArtificialSequence DNA is PNA 6 ctcttccttt ttc 13 7 67 PRT Artificial SequencePNA-NLS 7 Ala Glu Glu Ala Ala Glu Glu Ala Ala Glu Glu Ala Ala Glu GluAla 1 5 10 15 Ala Glu Glu Ala Ala Glu Glu Ala Ala Glu Glu Ala Ala GluGlu Ala 20 25 30 Ala Glu Glu Ala Ala Glu Glu Ala Gly Lys Pro Ser Ser AspAsp Glu 35 40 45 Ala Thr Ala Asp Ser Gln His Ser Thr Pro Pro Lys Lys LysArg Lys 50 55 60 Val Glu Asp 65 8 8 PRT Artificial Sequence Sequencecorresponding to the NLS of the SV40 virus large T antigen 8 Met Pro LysLys Lys Arg Lys Val 1 5 9 16 PRT Artificial Sequence Sequencecorresponds to a neutral hybrid consisting of the SV40 N LS and theN-terminal flanking region of the NLS from polyoma virus VP2 protein 9Met Glu Glu Asp Thr Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Leu 1 5 1015 10 7 PRT Artificial Sequence DNA is PNA; mixed peptide/PNA sequence10 Gly Gly Gly Gly Gly Gly Gly 1 5 11 5 DNA Artificial Sequence DNA isPNA; mixed peptide/PNA sequence 11 tttcc 5 12 10 PRT Artificial Sequencesequence corresponds to the NLS of the SV40 virus large T antigen 12 MetLys Pro Val Thr Leu Tyr Asp Val Ala 1 5 10 13 9 PRT Artificial Sequencesequence corresponds to a neutral hybrid consisting of the SV40 N LS andthe N-terminal flanking region of the NLS from polyoma virus VP2 protein13 Lys Pro Val Thr Leu Tyr Asp Val Ala 1 5 14 21 DNA Artificial Sequencelac-operator-sequence 14 aattgtgagc ggataacaat t 21 15 20 DNA ArtificialSequence lac-operator-sequence 15 aattgtgagc gctcacaatt 20

What is claimed is:
 1. A nuclear transport agent for transferring anucleic acid from cytoplasm into a nucleus of a eukaryotic cellcomprising a first module and a second module, wherein the first moduleis module A that binds specifically to a DNA molecule so as not to formcomplexes consisting of more than one DNA molecule, and wherein thesecond module is module B that comprises an extended nuclearlocalization signal having a charge thus preventing the second modulefrom mediating nonspecific binding of the nuclear transport agent to theDNA molecule.
 2. The nuclear transport agent of claim 1, wherein thefirst module specifically binds a sequence of the DNA molecule.
 3. Thenuclear transport agent of claim 1, wherein the first modulespecifically binds a terminal sequence of the DNA molecule.
 4. Thenuclear transport agent of claim 3, wherein the first modulespecifically binds covalently to the terminal sequence of the DNAmolecule.
 5. The nuclear transport agent of claim 1, wherein the firstmodule comprises a synthetic peptide, a protein, a peptide nucleic acid,or a recombinant protein that specifically binds to the DNA molecule. 6.The nuclear transport agent of claim 1, wherein the second modulecomprises an extended nuclear localization signal having a substantiallyneutral net charge, wherein the charge facilitates nuclear transport ofthe nucleic acid.
 7. The nuclear transport agent of claim 1, wherein thesecond module comprises an extended nuclear localization signalcomprising a nuclear localization signal and flanking amino acids havinga substantially negative charge.
 8. The nuclear transport agent of claim7, wherein the extended nuclear localization signal comprises from about2 to about 40 flanking amino acids.
 9. The nuclear transport agent ofclaim 8, wherein the extended nuclear localization signal comprises fromabout 4 to about 20 flanking amino acids.
 10. The nuclear transportagent of claim 1, wherein the second module further comprises a spacerconsisting of one or more peptide sequences or non-peptide sequences,wherein the sequences are external to the nuclear localization signal.11. The nuclear transport agent of claim 10, wherein the spacerseparates the nuclear localization signal from the first module.
 12. Thenuclear transport agent of claim 1, wherein the first module is aprotein that binds to DNA in a sequence specific manner and the secondmodule further comprises a non nuclear localization signal.
 13. Thenuclear transport agent of claim 12, wherein the non nuclearlocalization signal facilitates transport of the DNA molecule into orwithin the cell.
 14. A gene transfer system comprising a nucleartransport agent according to claim 1 and further comprising a cationiclipid, a peptide, a polyamine, or a cationic polymer.
 15. (A) An invitro method for transporting a DNA molecule into a nucleus of aeukaryotic cell comprising: transfecting the cell with the DNA moleculeand a nuclear transport agent of claim 1 and contacting the DNA moleculewith the nuclear transport agent, wherein the DNA transport to thenucleus is facilitated via the nuclear transport agent.
 16. The methodof claim 15, wherein the eukaryotic cell is a primary cell.